The present invention relates generally to the fields of medical microbiology and immunology. More particularly, the invention provides methods for inducing the production of interleukin-12 in peripheral blood mononuclear cells and, thereby, inducing a type 1/Th1 T cell immune response that is central to an effective cell-mediated immune response to intracellular pathogens or interferon xcex3-sensitive tumors.
Infection with the intracellular pathogen Mycobacterium tuberculosis (M. tuberculosis) continues to produce great morbidity and mortality throughout the world with 8 million new cases of tuberculosis and 3 million deaths occurring annually. Such creates an urgency to understand mechanisms of cell-mediated immunity (CMI) to the infection (Bloom and Murray, Science 257: 1055-1064, 1992). The spectrum of clinical outcomes after infection with M. tuberculosis is determined largely by the interaction of T-cells and monocyte/macrophages. Two functionally distinct subsets of T-cells modulate the outcome of such intracellular infections (Mosmann et al., J. Immunol 136:2348-2357, 1986). In human infectious disease, Type 1 T-cells that produce interleukin-2 (IL-2) and interferon xcex3 (IFN-xcex3) activate macrophages to kill or inhibit the growth of pathogens, resulting in mild or self-curing disease (Yamamura et al., Science 254:277-279, 1991; Salgame et al., Science 254:279-282, 1991). In contrast, type 2 T-cells which produce interleukins-4, -5, and -10 (IL-4, IL-5, IL-10) augment humoral responses (circulating antibody responses) and inhibit CMI, resulting in fulminant disease. The differentiation of naive T-cells into producing either type 1 or type 2 cytokine profiles is shaped by the cytokine milieu produced by the surrounding monocytes and macrophages. In particular, interleukin-12 (IL-12) is a key bridge between the innate immune response of monocyte/macrophages and the adaptive immune response of type 1 T-cells (Romagnani, S., Immunol Today 13:379-381, 1992).
IL-12, a cytokine or hormone-like substance, is produced by activated monocytes as a heterodimeric protein composed of p35 and p40 subunits. The p40 subunit is inducible and is considered to be the regulatory component for IL-12 expression (D""Andrea et al., J Exp Med 176:1387-1398, 1992). IL-12 production in response to microbial pathogens leads to activation of natural killer (NK) cells and T-cells with ensuing production of IFN-xcex3. In the presence of high IFN-xcex3 and low IL-4, there is subsequent development of a type 1 T-cell cytokine response and vigorous CMI (Hsieh et al., Science 260:547-549, 1993; Gazzinelli et al., J Immunol 153:2533-2543, 1994; Heinzel et al., J Exp Med 177:1505-1509, 1993).
In animal models of infection due to intracellular pathogens, the production of IL-12 is important in the generation of protective, Th1-mediated, immunity (Scott et al., J Exp Med 168:1675-1684, 1988; Heinzel et al.; J Exp Med 169:591989; Liew et al., Eur J Immunol 19:1227-1232, 1989; Sadick et al.,J Exp Med 171:115-127, 1990). For example, in mycobacterial infection, IL-12 production at the site of disease is a prominent characteristic of the resistant phenotype or self-limited disease. In tuberculosis, IL-12 has been found in the pleural fluid of patients with tuberculous pleuritis; and, anti-IL-12 antibodies partially inhibit the proliferative response of the pleural fluid lymphocytes to M. tuberculosis (Zhang et al., J Clin Invest 93:1733-1739, 1994). In leprosy, IL-12 induces the expansion of mycobacteria-reactive T-cells which produce IFN-xcex3, but has little effect on T-cells which produce IL-4 (Sieling et al., J Immunol 153:3639-3647, 1994). Further, in a murine model, exogenous administration of IL-12 increases the resistance of mice to M. tuberculosis infection via the IFN-xcex3 pathway (Flynn et al., J Immunol 155:2515-2524, 1995; Cooper et al., Immunology 84:423-432, 1995). In addition, production of IFN-xcex3 is required for immunity to mycobacterial infection (Flynn et al., J Exp Med 178:2249-2254, 1993; Cooper et al., J Exp Med 178:2243-2247, 1993).
A number of prokaryotic lipoproteins reportedly are potent macrophage stimulators. Examples include the TraT and Braun lipoproteins of E. coli, the outer membrane proteins of Treponema pallidum and Borrelia burgdorferi, and a lipoprotein of Mycoplasma fermentans. The B. burgdorferi OspA antigen and the 47 kDa antigen of T. pallidum have been reported to induce IL-12 mRNA (Ma et al., Infect Immun 62:3663, 1994; Radolf et al., J Immunol 154:2866, 1995).
The nature of an immune response reflects the profile of antigen-specific lymphocytes that are stimulated by the immunization. T cells consist of subpopulations that may be stimulated by different types of antigens and perform different effector functions. For example, in viral infections, viral antigens are synthesized in infected cells and presented in association with class I major histocompatibility complex (MHC) molecules leading to stimulation of CD8+, class I MHC-restricted cytotoxic lymphocytes. In contrast, extracellular microbial antigens are endocytosed by antigen presenting cells, processed, and presented in association with class II MHC molecules. This activates CD4+, class II MHC-restricted helper T cells, leading to antibody production and macrophage activation but relatively inefficient development of cytotoxic lymphocytes.
IL-12 promotes the development of Th1 cells, and microbes that stimulate macrophages to produce IL-12 or natural killer (NK) cells to produce IFN-xcex3 induce Th1-dominated responses. A type 1/Th1 T-cell response is where CD4+ helper Th1 cells secrete interleukin-2 and interferon-xcex3 which activates macrophages and are the principal effectors of cell-mediated immunity against intracellular microbes and of delayed type hypersensitivity. The antibody isotypes stimulated by Th1 cells are effective at activating complement and opsonizing antigens for phagocytosis. Therefore, Th1 cells trigger phagocyte-mediated host defense.
IL-4 stimulates differentiation of CD4+ T cells toward Th2 cells, and parasites, in general, induce early IL-4 production. Th2 cells also produce interleukin-4, which stimulates IgE antibody production, IL-5, IL-10 and IL-3, which together with IL-4 suppress cell-mediated immunity. Therefore, the Th2 subset of T cells is mainly responsible for phagocyte-independent host defense, e.g., against parasites; which is mediated by IgE and for allergic reactions, which are due to IgE-dependent activation of mast cells and basophils.
Protective immunity induced by vaccination is dependent on the capacity of the vaccine to elicit the appropriate immune response to either resist, control, or eliminate the pathogen. Adjuvants are substances capable of increasing the immunogenicity of antigens; such substances include aluminum salts, bacterial endotoxins, bacillus Calmette-GuJrin (BCG), or Bordetella pertussis, for example. Adjuvants stimulate the immune response by combining with antigen and forming an aggregate; this aggregate acts as a depot for prolonged antigen stimulation. In experiments with animals, the most frequently used adjuvants are Freund""s complete and incomplete adjuvants, which include water and oil emulsions with or without heat-killed Mycobacterium tuberculosis. The use of bacterial adjuvants to augment systemic immune responses in a nonspecific way is not without possible dangers. Repeated inoculation with live BCG organisms has caused systemic mycobacterial infections in several patients. To avoid this complication, other bacterial adjuvants, such as killed preparations of Corynebacterium parvum have been tested, and attempts have been made to extract the immunopotentiating component from BCG. In addition to prolonging antigen stimulation, adjuvants may also stimulate the immune response by influencing the cytokine milieu; activity and recruiting specific cell types, etc.
Responding to the need for identification of the active component of these adjuvants to solve such problems, the present inventors provide herein the characterization of mycobactenral lipoproteins that induce IL-12 production and that are useful in the development of new vaccine and therapeutic strategies.
The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing methods for inducing interleukin-12 production and inducing a type 1/Th1 T cell response, thereby stimulating cell-mediated immunity for prevention or treatment of pathogen infections or treatment of an interferon xcex3-sensitive tumor. The methods of the present invention are useful, by way of example, where IL-12-inducible peripheral blood mononuclear cells are in need of inducing to produce IL-12; in vivo where a subject, preferably a human patient, is in need of IL-12 induction to enhance the type 1/Th1 T cell response for enhanced cell-mediated immunity; and ex vivo where body fluids, such as blood or bone marrow, may be removed temporarily from a body and treated with compositions of the present invention for inducing IL-12 with resultant enhanced cell-mediated immunity.
Enhanced IL-12 production causes in vivo a type 1/Th1 T cell response which is required for cell-mediated immunity necessary in treatment or prevention of infections by microbial pathogens, intracellular or extracellular, as well as tumors that are interferon xcex3-sensitive.
One aspect of the invention provides a method of inducing a type 1/Th1 T-cell response in a subject. In one embodiment, the-method comprises administering to a subject a lipopeptide having an N-terminal ester- or amide-linked fatty acyl group in an amount effective to induce a type 1/Th1 T-cell response. A further embodiment is a method of inducing interleukin-12 in a subject comprising administering to the subject said lipopeptide in an amount effective to induce interleukin-12. One of skill in the art in light of the present disclosure can see that IL-12 induction is broader than the specific induction of cytotoxic T-cells. EP 0431327 reports the specific induction of cytotoxic T cells. The present invention proposes a conjugate compound that includes a consensus lipoprotein sequence plus an N-terminal portion of the M. tuberculosis 19 kDa antigen plus the protein or peptide of interest. This compound is post-transtationally modified in the appropriate manner to have activity (e.g., acylation and/or glycosylation). This conjugate compound is designed to induce a profound IL-12 release and a type 1 cell-mediated response; and to induce a specific pattern of NK activity, T-helper as well as T-cell cytotoxic responses.
The presently disclosed invention may be used in conjunction with an in vitro cell or cell culture of peripheral blood mononuclear cells having the capacity to produce IL-12; or with an animal having a functional immune system, especially a human patient in need of stimulation of the type 1/Th1 immune response. Such constitute definitions of a subject to be employed with the present inventive methods. The animal may be in need of a cell-mediated immune response to a pathogen, in particular, to the pathogen M. tuberculosis; or in need of a cell-mediated enhanced natural immunity to an interferon xcex3-sensitive tumor.
A peripheral blood mononuclear cell having the capacity to produce IL-12 is a mononuclear cell having a gene or part of a gene that encodes IL-12, and the regulatory genetic make-up and cellular machinery sufficient to respond to a lipopeptide of the present invention to induce the gene to produce messenger RNA, as well as cellular machinery sufficient to provide for the messenger RNA to be translated into the protein IL-12. A peripheral blood mononuclear cell includes a macrophage, monocyte, adherent cell, cell lines thereof, or the like.
A lipopeptide having an N-terminal ester- or amide-linked fatty acyl group has the capacity to induce IL-12 production when brought in contact with an IL-12-inducible PBMC. Capacity to induce IL-12 production means that upon exposure to the lipopeptide, the subject is stimulated to synthesize and produce the cytokine IL-12. An amount effective to induce the type 1/Th1 T-cell response means that the lipopeptide is administered in sufficient quantity so that interleukin-12 is produced which, in turn, activates T cells to produce interferon xcex3 which, in turn, activates cell-mediated immunity.
A method of protecting a subject against infection by M. tuberculosis by inducing a type 1/Th1 T cell response and a method for treating a human patient having a condition responsive to a type 1/Th1 T cell immune response are farther aspects of the invention. Each method comprises administering to the subject a lipopeptide having an N-terminal ester- or amide-linked fatty acyl group in an amount effective to induce the type 1/Th1 T-cell response. Protecting a subject means prevention by providing the lipopeptide in a vaccine prior to exposure to a pathogen, for example, or means treatment of a subject already having an infection or an interferon xcex3-sensitive tumor, for example.
A particular advantage of the present invention is the identification of the active agents of the mycobacterium cell wall having activity for inducing IL-12. Heretofore, it was necessary to administer the complete complement of normal or heat-killed cell wall components to induce an immune response. The present inventors"" showing that the 19 kDa lipoprotein of M. tuberculosis is the active agent for inducing IL-12 means that the active agents or active portions thereof are useful for any condition benefiting from administration of IL-12. Other advantages include the ability to stimulate IL-12 release by compounds not usually associated with type 1 T-cell response, or to switch a predominant type 2 response to a type 1 response using the proposed invention.
The present invention contributes towards the development of a new class of vaccines against deadly microbial pathogens by providing molecules and mechanisms that gerierate effective immunity. IL-12 has been identified as one such molecule that can serve as an adjuvant in vaccination against intracellular pathogens. However, the utility of recombinant IL-12 as an adjuvant in humans is at present unclear. The present invention shows that lipopeptides, especially mycobacterial lipopeptides, may present a more natural approach to the induction of cell-mediated immunity. Lipopeptides of the present invention may be used in a new generation of protein subunit vaccines.
Following long-standing patent law convention, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean xe2x80x9cone or morexe2x80x9d when used in this application, including the claims.
SDS-PAGE: Sodium dodecyl sulfatexe2x80x94polyacrylamide gel electrophoresis
ELISA: Enzyme-linked imnmunoadsorbent assay
PBMC: Peripheral blood mononuclear cells
LPS: Lipopolysaccharide
kDa: Kilodalton
CMI: Cell-mediated immunity
IL: Interleukin
IFN-xcex3: Interferon-xcex3